Post-translational modifications are essential regulators of in vivo protein function that increase biological complexity by effectively expanding the amino acid code from ~20 amino acids to several hundred building blocks. Our studies will focus on the enzymes that catalyze two post-translational modification pathways, prenylation and acetylation, where aberrant modifications have been implicated in the development of myriad pathological states, such as cancer, diabetes, aging, cardiovascular disease, neurological disorders and genetic diseases. As a result, acetylation and prenylation are validated targets for drug development. The majority of protein prenylation, whereby an isoprenoid group is appended near the C-terminus of a target protein, is catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). These modifications often target proteins to membranes, and are essential for the activity of many signal transduction pathways, such as Ras signaling pathways. After prenylation, the C-terminus can be further modified by proteolysis and carboxymethylation. One modifying enzyme, ZMPSTE24 is a zinc metalloprotease that cleaves the C-terminus of prenylated proteins, and mutations in this enzyme lead to the Hutchinson-Gilford progeria syndrome. Lysine acetylation is regulated by the relative activity of histone acetyl transferases that catalye protein lysine-N-acetylation, and histone deacetylases (HDACs) that catalyze hydrolysis of the N- acetyl-lysine modification. Acetylation regulates a plethora of cellular processes, and mutations in HDAC8 lead to the development of the Cornelia de Lange syndrome (CDLs). However, the full complement of in vivo substrates for these enzymes is unknown, and the functional basis for substrate selectivity is undefined. Moreover, the effects of these modifications on protein function and cellular pathways are frequently unclear. Here we propose to develop methods to answer significant questions concerning post-translational modification pathways, including the in vivo specificity and regulation of the individual enzymes. We intend to evaluate the biological function of the prenylation pathway by identifying sequences required for prenylation, membrane localization and proteolysis. We also propose to identify the substrate sets for HDAC8 and HDAC11 by measuring the reactivity with libraries of peptides and proteins. Additionally, we will explore the regulation of HDAC8 by phosphorylation, metal switching and complex formation and analyze the effects of mutations associated with the CDLs. Our long-term goals are: (1) to determine the catalytic mechanism and molecular basis of specificity for each of these post-translational modifying enzymes; and (2) to examine the extent, biological function and regulation of prenylation and acetylation in vivo. Overall, our studies will assist in targeted drug design by describing the specific interactions that dictate substrate specificity and by identifying novel regulatory pathways as targets for disease therapies.